Activation or detoxification of mutagenic and carcinogenic compounds in transgenic Drosophila expressing human glutathione S-transferase.

Sensitivity of transgenic Drosophila melanogaster with expression of a human gene encoding the glutathione S-transferase alpha subunit (GSTA1-1) to 1,2:5,6-dibenzanthracene (DBA) and 1,2-dichloroethane (DCE) was investigated in the somatic mutation and recombination test (SMART). We performed the same assay in control transgenic flies expressing the bacterial lacZ gene. Three types of transgenic Drosophila strains carrying GSTA1-1 were used: two transgenic strains homozygous for the second chromosome with a single-copy transgene insertion and one strain with two transgene insertions. Larvae carrying the lacZ gene were significantly more sensitive to genotoxic effects of DBA than those carrying three copies of the GSTA1-1 gene. The larvae with lacZ expression showed significantly lower sensitivity to DCE compared with those expressing GSTA1-1. Finally, a pretreatment with buthionine-sulphoximine (BSO) in experiment with DCE significantly decreased the frequency of mutation events in larvae with three GSTA1-1 copies in comparison with others.     

Genotoxic activation of hydrazine, two dialkylhydrazines, thiourea and ethylene thiourea in the somatic w/w+ assay of Drosophila melanogaster

Genotoxic activation of hydrazine (HZ), two symmetrical dialkylhydrazines, namely, 1,2dimethylhydrazine and 1,2-diethylhydrazine (SDMH and SDEH), thiourea (TU) and ethylene thiourea (ETU) has been evaluated by means of the w/w+ somatic assay of Drosophila. Both low bioactivation insecticide-susceptible (IS) and high biotransformation insecticide-resistant (IR) strains were used. The combined application of insecticide-susceptible and insecticide-resistant strains should, in principle, detect somatic cell recombinagens in the Drosophila melanogaster in vivo w/w+ assay. The IS strain was more susceptible to toxicity induced by the test chemicals than the IR stocks. Its performance in the biotransformation of the chemicals tested was rather poor. TU was inactive in all strains. With the active compounds, spot frequencies increased approximately linearly with dose for each spot type. SDEH gave a strong positive result in all three female genotypes exposed. HZ, ETU and SDMH were overall weakly positive in the IR strain Haag-79 (HG-R). Interestingly, ETU was clearly positive in the IR Hikone-R (HK-R) strain. A comparison of the recombinagenic potencies between the active and the weakly positive compounds, and among strains, showed pronounced genotype-dependent differences between the low and the high bioactivation strains. The ability of Drosophila to express several procarcinogens in relation to insecticide-resistance after activation catalyzed by CYP450 enzymes is discussed.     

Biodegradation of Phthalate Isomers by Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter lwoffii ISP4

Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter lwoffii ISP4 capable of utilizing phthalate isomers were isolated from the soil using enrichment culture technique. The strain ISP4 metabolizes isophthalate, while PPD and PP4 utilizes all three phthalate isomers (ortho-, iso- and tere-) as the sole carbon source. ISP4 utilizes isophthalate (0.1%) more rapidly (doubling time, 0.9 h) compared to PPD (4.64 h), PP4 (7.91 h) and other reported strains so far. The metabolic pathways in these isolates were initiated by dihydroxylation of phthalate isomers. Phthalate is hydroxylated to 3,4-dihydro-3,4-dihydroxyphthalate and 4,5-dihydro-4,5-dihydroxyphthalate in strains PP4 and PPD, respectively; while terephthalate is hydroxylated to 2-hydro-1,2-dihydroxyterephthalate. All three strains hydroxylate isophthalate to 4-hydro-3,4-dihydroxyisophthalate. The generated dihydroxyphthalates were subsequently metabolized to 3,4-dihydroxybenzoate (3,4-DHB) which was further metabolized by ortho ring-cleavage pathway. PP4 and PPD cells grown on phthalate, isophthalate or terephthalate showed respiration on respective phthalate isomer and the activity of corresponding ring-hydroxylating dioxygenase, suggesting the carbon source specific induction of three different ring-hydroxylating dioxygenases. We report, for the first time, the activity of isophthalate dioxygenase and its reductase component in the cell-free extracts. The enzyme showed maximum activity with reduced nicotinamide adenine dinucleotide (NADH) in the pH range 8–8.5. Cells grown on glucose failed to respire on phthalate isomers and 3,4-DHB and showed significantly low activities of the enzymes suggesting that the enzymes are inducible.     

The synthesis of 3,4-dideoxyhex-3-enitols

Syntheses of (E)-3,4-dideoxy-erythro-, (Z)-3,4-dideoxy-D-threo- and (E)-3,4-dideoxy-D-threo-hex-3-enitols are described. The action of potassium selenocyanate on 1,2:5,6-di-O-isopropylidene-D-mannitol 3,4-di-p-toluenesulfonate has been reexamined. Epoxidation of (E)-3,4-dideoxy-1,2:5,6-di-O-isopropylidene-D-threo-hex-3-enitol affords 3,4-anhydro-1,2:5,6-di-O-isopropylidene-D-mannitol and -D-iditol in the approximate proportions of 3:1. The configurations of the two epoxides were assigned on the basis of the reaction of the latter compound with sodium methoxide to give 1,2:5,6-di-O-isopropylidene-4-O-methyl-D-altritol.     

Chromosome aberrations in cultured central mudminnow heart cells and Chinese hamster ovary cells exposed to polycyclic aromatic hydrocarbons and sediment extracts

1. Genotoxicity experiments were conducted with cultured fish cells to determine if the high frequency of epidermal papillomas observed in lemon sole from Sturgeon Bank, where a sewage treatment plant discharges, could be correlated with contamination of the sediments with chemicals such as 3,4-benzopyrene. 2. The frequency of chromosome aberrations was measured in cultured Umbra limi heart (U1-H) and Chinese hamster ovary (CHO) cells following exposure to the polycyclic aromatic hydrocarbons (PAH) 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA), and pyrene (PY), activated using S9 prepared from rainbow trout liver. 3. An increase in the chromosome aberration frequency was only observed following exposure to fish S9-activated BP in both cell lines. 4. Following exposure of the cells to both Sturgeon Bank and Spanish Bank sediment extracts, it was determined that a higher level of toxic and genotoxic activity was associated with the Sturgeon Bank sediments. 5. Since the detection of PAH genotoxicity requires the presence of S9, and since a higher level of genotoxic activity was noted following sediment extract exposures with no S9 present, this suggests that the extracts contain a complex mix of chemicals, some of which express genotoxic activity. 6. An assessment using the micronucleus test failed to indicate in vivo genotoxicity in fish collected from Sturgeon and Spanish Banks. 7. It was, therefore, difficult to associate the observed sediment genotoxicity with the previously noted high incidence of epidermal papillomas in lemon sole from this area.     

Regio- and stereoselective cyclizations of dianhydro sugar alcohols catalyzed by a chiral (salen)Co(III) complex

The (salen)Co(III)OAc ((R,R)-1 and (S,S)-1) catalyzed cyclizations of the chiral dianhydro sugars, 1,2:5,6-dianhydro-3,4-di-O-methyl-D-glucitol (2), 1,2:5,6-dianhydro-3,4-di-O-methyl-D-mannitol (3), 1,2:5,6-dianhydro-3,4-di-O-methyl-L-iditol (4), and 1,2:4,5-dianhydro-3-O-methyl-L-arabinitol (5), is a facile method for the synthesis of anhydroalditol alcohols. Cyclization of 2 using (R,R)-1 and (S,S)-1 proceeded diastereoselectively to form 2,5-anhydro-3,4-di-O-methyl-D-mannitol (6) and 2,5-anhydro-3,4-di-O-methyl-L-iditol (7), respectively. The cyclization of 3 and 5 is a novel method for obtaining 1,6-anhydro-3,4-di-O-methyl-D-mannitol (11) and a stereoselective route to 1,5-anhydro-3-O-methyl-L-arabinitol (13). It is proposed that the reaction occurs via endo-selective cyclization of an epoxy alcohol produced by the endo-selective ring-opening of one of the two epoxide moieties in the starting material.     

CO binding studies of engineered cytochrome P-450ds: effects of mutations at putative distal sites in the presence of polycyclic hydrocarbons

The kinetic parameters of CO binding to genetically engineered cytochrome P-450d (P-450d) and two putative distal mutants, Glu318Asp and Thr322Ala, have been evaluated in the presence and absence of polycyclic hydrocarbons. The dissociation constant (Kd) of CO from wild-type P-450d was decreased by half (from 1.8 microM to approximately 0.9 microM) in the presence of phenanthrene or anthracene but was increased to 11 microM in the presence of 1,2:3,4-dibenzanthracene or 7,8-benzoflavone. These changed Kd values were not altered markedly by mutations at the putative distal site. In contrast, the recombination rate constants (kon) of CO to the Glu318Asp mutant in the presence of phenanthrene (15.5 X 10(5) M-1 s-1) and 7,8-benzoflavone (0.75 X 10(5) M-1 s-1) were much larger than those for the wild type. Similar but smaller increases of the kon values were observed for the Thr322Ala mutant. It was suggested that phenanthrene and anthracene distort the Fe-C-O bond and/or affect the access of CO to wild-type P-450d in an opposite way from 1,2:3,4-dibenzanthracene and 7,8-benzoflavone. Glu318 and Thr322 may be located so close to a CO binding channel in ferrous P-450d that mutations of these residues can open the sterically hindered CO channel caused by the hydrocarbons.     

Sugar orthocarbonates

A simple procedure is described for preparing sugar orthocarbonates. It is based on treating the corresponding thionocarbonate in pyridine with cupric acetate and an alcohol, such as methanol, ethanol, or isopropyl alcohol. Treatment of 1,2:5,6-di-O-isopropylidene-D-mannitol 3,4-thionocarbonate with diols, such as 1,2-ethanediol, 1,2-propanediol, or 1,2:5,6-di-O-isopropylidene-D-mannitol, also gave orthocarbonates. Methyl thionocarbonate, S-methyl xanthate, and dithiobis(thioformate) derivatives of 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose all gave the trimethyl orthocarbonate upon treatment with methanol in the presence of pyridine and cupric acetate. The structure of the orthocarbonates was proved by elemental analysis, n.m.r., and mass spectra, and by treatment with mild acid to form carbonates. Treatment of 1,2:5,6-di-O-isopropylidene-3-thio-D-altritol 3,4-thionocarbonate with methanol or ethanol gave the corresponding orthothiocarbonate, but on treatment with 1,2-ethanediol or with sodium ethoxide the 3,4-episulfide resulted.     

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

The formation of dihydrodiols in the chemical or enzymic oxidation of dibenz[a,c]anthracene, dibenz[a,h]-anthracene and chrysene.

The formation of trans-dihydrodiols from dibenz[a,c]anthracene, dibenz[a,h]anthracene and chrysene by chemical oxidation in an ascorbic acid-ferrous sulphate-EDTA system and by rat-liver microsomal fractions has been studied using a combination of thin-layer (TLC) and high pressure liquid chromatography (HPLC) to separate the mixtures of isomeric dihydrodiols. The 1,2- and 3,4-dihydrodiols of dibenz[a,c]anthracene, the 1,2-,3,4- and 5,6-dihydrodiols of dibenz[a,h]anthracene and the 1,2-, 3,4- and 5,6-dihydrodiols of chrysene were formed in chemical oxidations. These dihydrodiols were also formed when the three parent hydrocarbons were metabolized by rat-liver microsomal fractions and, in addition, dibenz[a,c]anthracene yielded the 10,11-dihydrodiol. The 1,2- and 3,4-dihydrodiols of dibenz[a,c]anthracene have not been reported previously either as metabolites of the hydrocarbon or as products of chemical syntheses and the 5,6-dihydrodiol of chrysene was not detected in earlier metabolic studies.     

The metabolism of benz[a]anthracene and dibenz[a,h]anthracene and their 5,6-epoxy-5,6-dihydro derivatives by rat-liver homogenates

1. Benz[a]anthracene was hydroxylated by rat-liver homogenates on the 3,4-,5,6- or 8,9-bond to yield phenols and dihydrodihydroxy compounds. Metabolic action at the 7- and 12-positions was also detected. 5,6-Epoxy-5,6-dihydrobenzanthracene was converted into a phenol that is probably 5-hydroxybenzanthracene and 5,6-dihydro-5,6-dihydroxybenzanthracene. Both substrates yielded a product that is probably S-(5,6-dihydro-6-hydroxy-5-benzanthracenyl)glutathione. 2. Dibenz[a,h]anthracene was hydroxylated by rat-liver homogenates to yield products that are probably 3- and 4-hydroxydibenzanthracene, 1,2-dihydro-1,2-dihydroxydibenzanthracene, 3,4-dihydro-3,4-dihydroxydibenzanthracene and 5,6-dihydro-5,6-dihydroxydibenzanthracene. There was no evidence for metabolic action at the 7- and 14-positions. 5,6-Epoxy-5,6-dihydrodibenzanthracene was converted into a phenol that is probably 5-hydroxydibenzanthracene and 5,6-dihydro-5,6-dihydroxydibenzanthracene. Both substrates yielded a glutathione conjugate that is probably S-(5,6-dihydro-6-hydroxy-5-dibenzanthracenyl)glutathione. 3. The synthesis of 5,6-epoxy-5,6-dihydrodibenzanthracene is described and the reactions of this epoxide and 5,6-epoxy-5,6-dihydrobenzanthracene with water and thiols have been investigated. 4. The oxidation of dibenzanthracene in the ascorbic acid-Fe(2+) ion-oxygen model system is described.     

Effect of different isomers of dihydroxybenzoic acids (DBA) on the rate of DL-dopa oxidation by mushroom tyrosinase.

Dihydroxybenzoic acids (DBA), such as 3,4-DBA, 3,5-DBA, and 2,4-DBA--at all concentrations tested--inhibited the rate of DL-DOPA oxidation to dopachrome (lambda max = 475 nm) by mushroom tyro0sinase. 2,3-DBA and 2,5-DBA at relatively low concentration had a synergistic effect on the reaction, whereas at relatively high concentrations they inhibited the rate of DL-DOPA oxidation. The synergistic effect of 0.6-13.3 mM 2,3-DBA on the rate of DL-DOPA oxidation to dopachrome (lambda max = 475 nm) was found to be due to the ability of 2,3-DBA-o-quinone (formed by the oxidation of 2,3-DBA by mushroom tyrosinase or by sodium periodate) to oxidize DL-DOPA to dopachrome (via dopaquinone) non-enzymatically. A similar explanation is likely to be valid for the synergism exerted by 2,5-DBA on the rate of DL-DOPA oxidation by mushroom tyrosinase.     

Syntheses and reactions of 5-O-acetyl-1,2-anhydro-3-O-benzyl-alpha-D-ribofuranose and beta-D-lyxofuranose, 5-O-acetyl-1,2-anhydro-3,6-di-O-benzyl- and 1,2-anhydro-5,6-di-O-benzoyl-3-O-benzyl-beta-D-mannofuranose, and 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-alpha-D-glucopyranose and -beta-D-talopyranose

The title compounds 5-O-acetyl-1,2-anhydro-3-O-benzyl-alpha-D-ribofuranose and 5-O-acetyl-1,2-anhydro-3-O-benzyl-beta-D-lyxofuranose, and 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-alpha-D-glucopyranose and 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-beta-D-talopyranose, and 5-O-acetyl-1,2-anhydro-3,6-di-O-benzyl-beta-D-mannofuranose and 1,2-anhydro-5,6-di-O-benzoyl-3-O-benzyl-beta-D-mannofuranose have each been synthesized from the corresponding 2-O-tosylate and 1-free hydroxyl intermediates by base-initiated intramolecular S(N)2 ring closure in almost quantitative yields. Acetyl and benzoyl groups were not affected in the ring closure reactions. Condensation of 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-alpha-D-glucopyranose and 5-O-acetyl-1,2-anhydro-3,6-di-O-benzyl-beta-D-mannofuranose with 1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose in the presence of ZnCl2 as the catalyst afforded the 1,2-trans-linked 6-O-acetyl-3,4-di-O-benzyl-beta-D-glucopyranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose and 5-O-acetyl-3,6-di-O-benzyl-alpha-D-mannofuranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose as the sole products in satisfactory yields, while condensation of 5-O-acetyl-1,2-anhydro-3-O-benzyl-beta-D-lyxofuranose with 3-O-benzyl-1,2-O-isopropylidene-alpha-D-xylofuranose yielded the 1,2-trans-linked 5-O-acetyl-3-O-benzyl-alpha-D-lyxofuranosyl-(1-->5)-3-O-benzyl-1,2-O-isopropylidene-alpha-D-xylofuranose as the sole product in a good yield. The 6-O-acetyl group in the glycosyl donor, 6-O-acetyl-1,2-anhydro-3,4-di-O-benzyl-alpha-D-glucopyranose, did not influence the stereoselectivity of the ring-opening-coupling reaction.     

Effect of Different Isomers of Dihydroxybenzoic Acids (DBA) on the Rate of DL-DOPA Oxidation by Mushroom Tyrosinase

Dihydroxybenzoic acids (DBA), such as 3,4-DRA, 3,5-DBA, and 2,4-DBA—at all concentrations tested—inhibited the rate of DL-DOPA oxidation to dopachrome (λmax = 475 nm) by mushroom tyrosinase. 2,3-DBA and 2,5-DBA at relatively low concentration had a synergistic effect on the reaction, whereas at relatively high concentrations they inhibited the rate of DL-DOPA oxidation. The synergistic effect of 0.6-13.3 mM 2,3-DRA on the rate of DL-DOPA oxidation to dopachrome (λmax = 475 nm) was found to be due to the ability of 2,3-DBA-o-quinone (formed by the oxidation of 2,3-DBA by mushroom tyrosinase or by sodium periodate) to oxidize DL-DOPA to dopachrome (via dopaquinone) non-enzymatically. A similar explanation is likely to be valid for the synergism exerted by 2,5-DBA on the rate of DL-DOPA oxidation by mushroom tyrosinase.     

Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans."In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer.     

Difference in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the Salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzoflavone).

In the enzymic assay with PCBs-induced microsomes, BP hydroxylation revealed a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by PB plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed.

In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations.

In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors.

From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.     

Mutagenicity of stereochemical configurations of 1,2-epoxybutene and 1,2:3,4-diepoxybutane in human lymphblastoid cells

The carcinogenicity of 1,3-butadiene (BD) is related to its bioactivation to several DNA-reactive metabolites; accumulating evidence suggests that the stereochemistry of these BD intermediates may play a significant role in the mutagenic and carcinogenic actions of the parent compound. The objective of this study was to evaluate the cytotoxicity and mutagenicity of stereochemical forms of 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB), two genotoxic BD metabolites, in a human lymphoblastoid cell line, TK6. Cytotoxicity was measured by comparing cloning efficiencies in chemical-exposed cells versus those in control cells. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK) mutant frequencies (MFs) were measured using a cell cloning assay. HPRT mutants collected from cells exposed to the three forms of DEB were analyzed by PCR to characterize large genetic alterations. All the three stereoisomers of DEB caused increased HPRT and TK MFs compared to the concurrent control samples. There were no significant differences in cytotoxicity or mutagenicity among the three isomers of DEB in TK6 cells. Molecular analysis of HPRT mutants revealed similar distributions of types of mutations among the three isomers of DEB. There were also no statistically significant differences in mutagenic efficiencies between the two isomers of EB in TK6 cells. These results were consistent with the in vivo findings that there was little difference in the mutagenic efficiencies of racemic-DEB versus meso-DEB in rodents. Thus, in terms of mutagenic efficiency, stereochemical configurations of EB and DEB are not likely to play a significant role in the mutagenicity and carcinogenicity of BD.     

Degradation of phenanthrene by <Emphasis Type="Italic">Burkholderia</Emphasis> sp. C3: initial 1,2- and 3,4-dioxygenation and <Emphasis Type="Italic">meta</Emphasis>- and <Emphasis Type="Italic">ortho</Emphasis>-cleavage of naphthalene-1,2-diol

Burkholderia sp. C3 was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated site in Hilo, Hawaii, USA, and studied for its degradation of phenanthrene as a sole carbon source. The initial 3,4-C dioxygenation was faster than 1,2-C dioxygenation in the first 3-day culture. However, 1-hydroxy-2-naphthoic acid derived from 3,4-C dioxygenation degraded much slower than 2-hydroxy-1-naphthoic acid derived from 1,2-C dioxygenation. Slow degradation of 1-hydroxy-2-naphthoic acid relative to 2-hydroxy-1-naphthoic acid may trigger 1,2-C dioxygenation faster after 3 days of culture. High concentrations of 5,6-␣and 7,8-benzocoumarins indicated that meta-cleavage was the major degradation mechanism of phenanthrene-1,2- and -3,4-diols. Separate cultures with 2-hydroxy-1-naphthoic acid and 1-hydroxy-2-naphthoic acid showed that the degradation rate of the former to naphthalene-1,2-diol was much faster than that of the latter. The two upper metabolic pathways of phenanthrene are converged into naphthalene-1,2-diol that is further metabolized to 2-carboxycinnamic acid and 2-hydroxybenzalpyruvic acid by ortho- and meta-cleavages, respectively. Transformation of naphthalene-1,2-diol to 2-carboxycinnamic acid by this strain represents the first observation of ortho-cleavage of two rings-PAH-diols by a Gram-negative species.     

Degradation of 2,5-dichlorobenzoic acid byPseudomonas aeruginosa JB2 at low oxygen tensions

From long-term chemostat experiments, variants ofPseudomonas aeruginosa JB2 were obtained which exhibited altered properties with respect to the metabolism of 2,5-dichlorobenzoic acid (2,5-DBA). Thus, unlike the original strain JB2-WT, strain JB2-var1 is able to grow in continuous culture on 2,5-DBA as the sole limiting carbon and energy source. Yet, at a dilution rate of 0.07 h^–1 and a dissolved oxygen concentration of 12 µM, even with this strain no steady states with 2,5-DBA alone could be established in continuous cultures. Yet another strain was obtained after prolonged continuous growth of JB2-var1 in the chemostat. It has improved 2,5-DBA degrading capabilities which become apparent only during growth in continuous culture: a lower apparent K _m for 2,5-DBA and lowered steady-state residual concentrations of 2,5 DBA. Although with this strain steady states were obtained at oxygen concentrations as low as 11 µM, at further lowered concentrations this was no longer possible. In C-limited continuous cultures of JB2-var1 or JB2-var2, addition of benzoic acid (BA) to the feed reduced the amounts of 2,5-DBA degraded, which was most apparent at low oxygen concentrations (< 30 µM). At higher dissolved oxygen concentrations the addition of BA resulted in increasing cell-densities but did not affect the residual steady state concentration of 2,5-DBA. Indeed, whole cell suspensions from chemostat cultures grown on BA plus 2,5-DBA did show a lower apparent affinity for 2,5-DBA than those from cultures grown on 2,5-DBA alone. These results indicate that in environments with low oxygen concentrations and alternative, more easily degradable, substrates the degradation rates of chloroaromatic compounds by aerobic organisms may be negatively affected.Abbreviations BA benzoic acid - 2,5-DBA 2,5-dichlorobenzoic acid - QO ₂ ^max maximum specific respiration rate     

Nucleosides LXXXIII. Synthetic studies on nucleoside antibiotics. 11. Synthesis of methyl 4-amino-3,4-dideoxy-β-D-ribo-hexopyranoside and -hexopyranosiduronic acid (derivatives related to the carbohydrate moiety of gougerotin)

Methyl 4-amino-3,4-dideoxy-β-D-ribo-hexopyranoside (17) and its uronic acid (19) were synthesized via a series of reactions starting from 1,2:5,6-di-O-isopropylidene-3-O-tosyl-α-D-glucofuranose. A method suitable for the large scale preparation of 3,4-dideoxy- 1,2:5,6-di-O-isopropylidene-α-D-erythro-hex-3-enofuranose(2) was devised.